Combined measurement of anti-CCP and serum amyloid A to assess rheumatoid arthritis

ABSTRACT

The present invention relates to a method aiding in the assessment of rheumatoid arthritis. The method especially is used in assessing the absence or presence of rheumatoid arthritis in vitro. It can be best practiced by analyzing biochemical markers, comprising measuring in a sample the concentration of anti-CCP and serum amyloid A and correlating the concentrations determined to the absence or presence of rheumatoid arthritis. To further improve the assessment of RA in a method of this invention the level of one or more additional marker may be determined together with anti-CCP and serum amyloid A and be correlated to the absence or presence of RA. The invention also relates to the use of a marker panel comprising anti-CCP and serum amyloid A in the diagnosis of rheumatoid arthritis and it teaches a kit for performing the method of the invention.

RELATED APPLICATIONS

This application is a continuation of PCT/EP2005/001861 filed Feb. 23,2005 and claims priority to EP 04004586.6 filed Feb. 27, 2004.

FIELD OF THE INVENTION

The present invention relates to a method aiding in the assessment ofrheumatoid arthritis. The method especially is used in assessing theabsence or presence of rheumatoid arthritis in vitro. It can be bestpracticed by analyzing biochemical markers, comprising measuring in asample the concentration of anti-CCP (cyclic citrullinated peptides) andserum amyloid A and correlating the concentrations determined to theabsence or presence of rheumatoid arthritis. To further improve theassessment of RA in a method of this invention the level of one or moreadditional marker may be determined together with anti-CCP and serumamyloid A and be correlated to the absence or presence of RA. Theinvention also relates to the use of a marker panel comprising anti-CCPand serum amyloid A in the diagnosis of rheumatoid arthritis and itteaches a kit for performing the method of the invention.

BACKGROUND OF THE INVENTION

Rheumatoid arthritis (“RA”) is a chronic, inflammatory, systemic diseasethat produces its most prominent manifestations in affected joints,particularly those of the hands and feet. The onset of rheumatoidarthritis can occur slowly, ranging from a few weeks to a few months, orthe condition can surface rapidly in an acute manner.

RA has a worldwide distribution and involves all ethnic groups. Althoughthe disease can occur at any age, the prevalence increases with age andthe peak incidence is between the fourth and sixth decade. Theprevalence estimates for the North American population vary from 0.3% to1.5%. Today, over 2,500,000 individuals are diagnosed with rheumatoidarthritis in the United States alone, with some statistics indicatingfrom 6.5 to 8 million potentially afflicted with the disease. Women areaffected 2-3 times more often than men.

The early symptoms of rheumatoid arthritis are mostly joint specificsuch as painful joints with joint swelling or tenderness, but may alsoinclude rather non-specific manifestations like stiffness, fever,subcutaneous nodules, and fatigue. Very characteristic is the symmetricinvolvement of joints. The joints of the hands, feet, knees and wristsare most commonly affected, with eventual involvement of the hips,elbows and shoulders. As the disease progresses, any type of motionbecomes very painful and difficult leading eventually to a loss offunction of the involved joints The more severe cases of rheumatoidarthritis can lead to intense pain and joint destruction. Some 300,000bone and joint replacement surgical procedures are performed annually inan effort to alleviate the pain and mobility loss resultant fromarthritis related joint destruction.

The most widely used system to classify RA is the American College ofRheumatology 1987 revised criteria for the classification of RA. (ArnettF C, et al., Arthritis Rheum 31 (1988) 315-324: “The American RheumatismAssociation 1987 revised criteria for the classification of rheumatoidarthritis.”). According to these criteria (known as ARA-criteria), apatient is said to have RA if the patient satisfies at least four of thefollowing seven criteria, wherein criteria 1-4 must be present for atleast six weeks: 1) morning stiffness for at least one hour, 2)arthritis of three or more joint areas, 3) arthritis of hand joints, 4)symmetrical arthritis, 5) rheumatoid nodules, 6) serum rheumatoid factor(“RF”), and 7) radiographic changes. These criteria have a sensitivityand specificity of approximately 90%.

The only biochemical marker generally accepted (see the aboveARA-criteria) and aiding in the diagnosis of RA is the rheumatoid factor(RF) as detected in serum.

The histological changes in RA are not disease-specific but largelydepend on the organ involved. The primary inflammatory joint lesioninvolves the synovium. The earliest changes are injury to the synovialmicrovasculature with occlusion of the lumen, swelling of endothelialcells, and gaps between endothelial cells, as documented by electronmicroscopy. This stage is usually associated with mild proliferation ofthe superficial lining cell layer. Two cell types constitute thesynovial lining: bone marrow derived type A synoviocyte, which hasmacrophage features, and mesenchymal type B synoviocyte. Both cell typescontribute to synovial hyperplasia, suggesting a paracrine interactionbetween these two cell types. This stage of inflammation is associatedwith congestion, oedema, and fibrin exudation. Cellular infiltrationoccurs in early disease and initially consists mainly of T lymphocytes.As a consequence of inflammation, the synovium becomes hypertrophic fromthe proliferation of blood vessels and synovial fibroblasts and frommultiplication and enlargement of the synovial lining layers.

Granulation tissue extends to the cartilage and is known as pannus. Thetissue actively invades and destroys the periarticular bone andcartilage at the margin between synovium and bone, known as erosive RA.

The articular manifestations of RA can be placed in two categories:reversible signs and symptoms related to inflammatory synovitis andirreversible structural damage caused by synovitis. This concept isuseful not only for staging disease and determining prognosis but alsofor selecting medical or surgical treatment. Structural damage in thetypical patient usually begins sometime between the first and secondyear of the disease (Van der Heijde, D. M., Br J Rheumatol 34 (1995)74-8). Although synovitis tends to follow a fluctuating pattern,structural damage progresses as a linear function of the amount of priorsynovitis.

The aetiology of the early events in RA remains elusive. An autoimmunecomponent is widely accepted today but other factors are still disputed.The possibility of a bacterial or viral infection has been vigorouslypursued. All efforts to associate an infectious agent with RA byisolation, electron microscopy, or molecular biology have failed. It ispossible that there is no single primary cause of RA and that differentmechanisms may lead to the initial tissue injury and precipitatesynovial inflammation.

Clinical signs of synovitis may be subtle and are often subjective.Warm, swollen, obviously inflamed joints are usually seen only in themost active phases of inflammatory synovitis. Cartilage loss and erosionof periarticular bone are the characteristic features of structuraldamage. The clinical features related to structural damage are marked byprogressive deterioration functionally and anatomically. Structuraldamage to the joint is irreversible and additive.

The effective treatment of rheumatoid arthritis has generally compriseda combination of medication, exercise, rest and proper joint protectiontherapy. The therapy for a particular patient depends on the severity ofthe disease and the joints that are involved. Non-steroidalanti-inflammatory drugs, corticosteroids, gold salts, methotrexate andsystemic immunosuppressants are widely used to reduce inflammation andjoint destruction. The use of steroids and immunosuppressants, however,has significant risks and side effects both in terms of toxicity andvulnerability to potentially lethal conditions. More recentlytherapeutics based on “biologicals” have been introduced intoRA-therapy. Such therapeutics, e.g., are soluble receptors or antibodiesdirected against TNF-

that significantly reduce inflammation. Though very promising,biologicals are still in limited use due to high costs.

Data from longitudinal clinical and epidemiologic studies provideguidelines for treatment. These studies emphasize 1) the need for earlydiagnosis, 2) identification of prognostic factors, and 3) earlyaggressive treatment. Earlier diagnosis and treatment, preferably withinthe first several months after onset of symptoms, may help preventirreversible joint damage.

SUMMARY OF THE INVENTION

Hence a need for methods, especially based on biochemical parameters,aiding in the assessment of rheumatoid arthritis exists. The presentinvention provides such methods and reagents for assessing the absenceor presence of rheumatoid arthritis in vitro. The methods will also aidin monitoring the efficacy of treatment in patients suffering from RA.

The present invention relates to a method for assessing the absence orpresence of rheumatoid arthritis in a patient comprising (a) measuringin a sample from the patient the concentration of anti-CCP and serumamyloid A, and (b) correlating the concentrations determined in step (a)to the absence or presence of rheumatoid arthritis in the patient.

The present invention further relates to a method for diagnosingrheumatoid arthritis in a patient comprising (a) measuring in a samplefrom the patient the concentration of anti-CCP and serum amyloid A, and(b) correlating the concentrations determined in step (a) to a diagnosisof rheumatoid arthritis in the patient.

The present invention further relates to a kit for assessing the absenceor presence of rheumatoid arthritis in a patient comprising reagents formeasuring anti-CCP in a patient sample and reagents for measuring serumamyloid A in a patient sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: ROC-analysis of patients diagnosed with RA versus controls incl.OA using log total RF alone.

FIG. 2: ROC-analysis of patients diagnosed with RA versus controls incl.OA using log anti-CCP alone.

FIG. 3: ROC-analysis of patients diagnosed with RA versus controls incl.OA using the combination log anti-CCP plus log SAA.

FIG. 4: ROC-analysis of patients diagnosed with RA versus controls incl.OA using the combination log anti-CCP plus log SAA plus log hyaluronicacid.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is directed to a method for assessing rheumatoidarthritis in vitro by biochemical markers, comprising measuring in asample the concentration of anti-CCP and serum amyloid A and correlatingthe concentrations determined to the absence or presence of rheumatoidarthritis.

The present invention also relates to the use of a marker panelcomprising at least anti-CCP and serum amyloid A in the diagnosis of RA.

The present invention also provides a kit for performing the methodaccording to the present invention comprising at least the reagentsrequired to specifically measure anti-CCP and serum amyloid A,respectively, and optionally auxiliary reagents for performing themeasurement.

In a first preferred embodiment the present invention relates to amethod for assessing rheumatoid arthritis in vitro by biochemicalmarkers, comprising measuring in a sample the concentration of anti-CCPand serum amyloid A and correlating the concentrations determined to theabsence or presence of rheumatoid arthritis.

As used herein, each of the following terms has the meaning associatedwith it in this section.

The articles “a” and “an” are used herein to refer to one or to morethan one (i.e. to at least one) of the grammatical object of thearticle. By way of example, “a marker” means one marker or more than onemarker.

The term “marker” or “biochemical marker” as used herein refers tomolecules to be used as targets for analyzing patient test samples.Examples of such molecular targets are proteins or polypeptidesthemselves as well as antibodies present in a sample. Proteins orpolypeptides used as a marker in the present invention are contemplatedto include any variants of said protein as well as fragments of saidprotein or said variant, in particular, immunologically detectablefragments. One of skill in the art would recognize that proteins whichare released by cells or present in the extracellular matrix whichbecome damaged, e.g., during inflammation could become degraded orcleaved into such fragments. Certain markers are synthesized in aninactive form, which may be subsequently activated by proteolysis. Asthe skilled artisan will appreciate, proteins or fragments thereof mayalso be present as part of a complex. Such complex also may be used as amarker in the sense of the present invention. Variants of a markerpolypeptide are encoded by the same gene, but differ in their PI or MW,or both (e.g., as a result of alternative mRNA or pre-mRNA processing,e.g. alternative splicing or limited proteolysis) and in addition, or inthe alternative, may arise from differential post-translationalmodification (e.g., glycosylation, acylation, and/or phosphorylation).

The term marker as indicated above according to the present inventionalso relates to antibodies present in a sample. In the case of RA theseantibodies are autoantibodies, i.e. antibodies in a patient sample whichbind to an antigen present in or on or produced by the patient's owncells.

The term “sample” as used herein refers to a biological sample obtainedfor the purpose of evaluation in vitro. In the methods of the presentinvention, the sample or patient sample preferably may comprise any bodyfluid. Preferred test samples include blood, serum, plasma, urine,saliva, and synovial fluid. Preferred samples are whole blood, serum,plasma or synovial fluid, with plasma or serum being most preferred.

The term “assessing rheumatoid arthritis” is used to indicate that themethod according to the present invention will (together with othervariables, e.g., the criteria set forth by the ARA (see above)) aid thephysician to establish his diagnosis of RA. In a preferred embodimentthis assessment will relate to the presence or absence of RA. As theskilled artisan will appreciate no biochemical marker is diagnostic with100% specificity and at the same time 100% sensitivity for a givendisease, rather biochemical markers are used to assess with a certainlikelihood or predictive value the presence or absence of a disease.Preferably the method according to the present invention aids inassessing the presence or absence of RA.

As the skilled artisan will appreciate the step of correlating a markerlevel to the presence or absence of RA can be performed and achieved indifferent ways. In general a reference population is selected and anormal range established. It is no more than routine experimentation, toestablish the normal range for both anti-CCP as well as serum amyloid Ausing an appropriate reference population. It is generally accepted thatthe normal range to a certain but limited extent depends on thereference population in which it is established. The ideal referencepopulation is high in number, e.g., hundreds to thousands, and matchedfor age, gender and optionally other variables of interest. The normalrange in terms of absolute values, like a concentration given, alsodepends on the assay employed and the standardization used in producingthe assay.

The levels for anti-CCP and serum amyloid A have been measured andestablished with the assay procedures given in the examples section. Ithas to be understood that different assays may lead to different cut-offvalues, without departing from the scope of the present invention.

Citrullinated peptides are antigens for rather important autoantibodiesas found in the sera of patients with RA. They have been intensivelystudied during the past years by several groups of researchers (cf.e.g., WO 98/08946; WO 98/22503;

WO 99/28344; WO 99/35167, WO 01/46222, and WO 03/050542). RecentlySchellekens and co-workers (Schellekens, G. A. et al., Arthritis Rheum.43 (2000) 155-163) reported that an ELISA-test based on specific cycliccitrullinated peptides (CCP) showed superior performance characteristicswith regard to diagnostic accuracy for RA as compared to the same assayusing linear peptides.

Auto-antibodies against CCP, i.e., antibodies which most likely arereactive with citrullinated polypeptides circulating in a patient serumand which bind to CCP in an in vitro assay are termed “anti-CCP”. Thepatent application of van Venroji et al. (WO 98/22503) describes certaincitrullinated peptides and shows that cyclization leads to an improvedreactivity of the respective peptides. In a specific example it is shownthat, if a peptide of the general formula HQCHQESTXGRSRGRCGRSGS (SEQ IDNO: 1), where X stands for citrulline, is cyclisized by a disulfide bondbetween the two cysteine residues, the sensitivity is increased to 63%as compared to 36% to the corresponding linear peptide. Asautoantibodies in patient sera have slightly different reactivity todifferent cyclic peptides a combination of peptides was suggested in WO98/22503 to further improve the assay.

In a preferred embodiment anti-CCP is measured as described by vanVenroij et al in WO 03/050542. In brief, a combination of peptides thatcontain epitope sites with the general formula X-G and X-nonG wherein Xstands for citrulline, G for glycine and nonG for any of the amino acidsH, I, W, S, R, K, Y, M, F, V, P, Cit or an analogue thereof is used toassess the level of anti-CCP antibodies (anti-CCP) in a sample. Specificpeptides useful in such assessment are disclosed in WO 03/050542. As theskilled artisan will readily appreciate, further improvements andrefinements regarding the cyclic citrullinated peptide antigen used inan assay to measure anti-CCP are possible which will e.g. result in analtered sequence of the cyclic citrullinated peptide sequence. However,such modifications will not depart from the spirit of this invention.

The antibody binding to CCP, i.e., anti-CCP, is measured in aserological assay. Preferably such assay is set up by using one or moreCCP as antigen and detecting the binding of anti-CCP antibodiescomprised in a sample to the CCP antigen by appropriate means.

Preferred means of detection are specific binding assays, especiallyimmunoassays. Immunoassays are well known to the skilled artisan.Methods for carrying out such assays as well as practical applicationsand procedures are summarized in related textbooks. Examples of relatedtextbooks are Tijssen, P., In: Practice and theory of enzymeimmunoassays, eds. R. H. Burdon and v. P. H. Knippenberg, Elsevier,Amsterdam, 1990, pp. 221-278) and various volumes of Methods inEnzymology, eds. S. P. Colowick, N. O. Caplan and S. P., Academic Press,1980), dealing with immunological detection methods, especially volumes70, 73, 74, 84, 92 and 121.

Anti-CCP antibodies may be detected by homogeneous assays formats, e.g.,by agglutination of latex particles coated with CCP.

Preferably a heterogeneous immunoassay is used to measure anti-CCP. Suchheterogeneous measurement is based on directly or indirectly coating CCPto a solid phase, incubating the solid phase with a sample known orsuspected to comprise anti-CCP antibodies under conditions allowing forbinding of anti-CCP antibodies to CCP, and directly or indirectlydetecting the anti-CCP antibody bound. A further assay format is theso-called double antigen bridge assay, wherein, in case of an anti-CCPmeasurement, CCPs are used both at the solid phase side as well as atthe detection side of this immunoassay and the autoantibodies in apatient sample form a bridge between these “double” antigens. Wherenecessary or appropriate, washing steps are included while performing aheterogeneous immunoassay.

Serum amyloid A (=SAA) is an acute phase protein of low molecular weightof 11.7 kDa. It is predominantly synthesized by the liver in response toIL-1, IL-6 or TNF-□ stimulation and is involved in the regulation of theT-cell dependent immune response. Upon acute events the concentration ofSAA increases up to 1000-fold reaching one milligram per milliliter. Itis used to monitor inflammation in diseases as divers as cysticfibrosis, renal graft refection, trauma or infections (Mozes, G. et al.,J Trauma 29 (1989) 71-74: “Serum amyloid A: An extremely sensitivemarker of tissue damage in trauma patients and indicator of acuteresponse in various diseases”). In rheumatoid arthritis it has incertain cases been used as a substitute for CRP, but, SAA is not yet aswidely accepted (Chambers R. E. et al., Ann. Rheum. Dis. 42 (1983)665-667: “Serum amyloid-A protein concentration in rheumatoid arthritisand its role in monitoring disease activity”).

Whereas for anti-CCP the (auto-)antibodies as comprised in a sample aremeasured, for SAA it is the marker molecule SAA itself, which isdetected. SAA for example can be measured by a competitive type or asandwich type immunoassay. SAA preferably is measured in a sandwichimmunoassay which is essentially based on an antibody specificallybinding to SAA which is directly or indirectly bound or capable ofbinding to a solid phase, an antibody specifically binding to SAA whichis detectably labeled, and incubating these reagents under conditionsallowing for binding of the anti-SAA antibodies to SAA in a sample,separating unbound detectably labeled antibody, determining the amountof labeled antibody bound via SAA, and correlating the amount of labeledantibody bound to the concentration of SAA in the sample.

The ideal scenario for diagnosis would be a situation wherein a singleevent or process would cause the respective disease as, e.g., ininfectious diseases. In all other cases correct diagnosis can be verydifficult, especially when the etiology of the disease is not fullyunderstood as is the case for RA. Therefore, generally various clinicalsymptoms and biological markers are considered together for diagnosis ofRA. Markers can either be determined individually or in a preferredembodiment of the invention they can be measured simultaneously using achip or a bead based array technology. The concentrations of thebiomarkers are then interpreted independently using an individualcut-off for each marker or they are combined for interpretation. It is apreferred embodiment of the invention to use an optimized multivariatecut-off for the underlying combination of biological markers and todiscriminate state A from state B e.g. diseased from healthy. In thistype of analysis the markers are no longer independent but form a markerpanel. It now could be established that combining the measurements ofanti-CCP and of SAA does improve the diagnostic accuracy for RA ascompared to either healthy controls or, also assessed, as compared topatients with osteoarthritis (OA). Especially the later finding is ofgreat importance, because patients with OA and RA, respectively, mayrequire quite different treatments.

As shown in the examples section the mere combination of the two markersanti-CCP and SAA significantly improves the diagnostic accuracy for RA.

Accuracy of a diagnostic method is best described by itsreceiver-operating characteristics (ROC). See especially Zweig, M. H.,and Campbell, G., Clin. Chem. 39 (1993) 561-577. The ROC graph is a plotof all of the sensitivity/specificity pairs resulting from continuouslyvarying the decision thresh-hold over the entire range of data observed.

The clinical performance of a laboratory test depends on its diagnosticaccuracy, or the ability to correctly classify subjects into clinicallyrelevant subgroups. Diagnostic accuracy measures the test's ability tocorrectly distinguish two different conditions of the subjectsinvestigated. Such conditions are for example health and disease orbenign versus malignant disease.

In each case, the ROC plot depicts the overlap between the twodistributions by plotting the sensitivity versus 1—specificity for thecomplete range of decision thresholds. On the y-axis is sensitivity, orthe true-positive fraction [defined as (number of true-positive testresults)/(number of true-positive+number of false-negative testresults)]. This has also been referred to as positivity in the presenceof a disease or condition. It is calculated solely from the affectedsubgroup. On the x-axis is the false-positive fraction, or 1—specificity[defined as (number of false-positive results)/(number oftrue-negative+number of false-positive results)]. It is an index ofspecificity and is calculated entirely from the unaffected subgroup.Because the true- and false-positive fractions are calculated entirelyseparately, by using the test results from two different subgroups, theROC plot is independent of the prevalence of disease in the sample. Eachpoint on the ROC plot represents a sensitivity/specificity paircorresponding to a particular decision threshold. A test with perfectdiscrimination (no overlap in the two distributions of results) has anROC plot that passes through the upper left corner, where thetrue-positive fraction is 1.0, or 100% (perfect sensitivity), and thefalse-positive fraction is 0 (perfect specificity). The theoretical plotfor a test with no discrimination (identical distributions of resultsfor the two groups) is a 45° diagonal line from the lower left corner tothe upper right corner. Most plots fall in between these two extremes.(If the ROC plot falls completely below the 45° diagonal, this is easilyremedied by reversing the criterion for “positivity” from “greater than”to “less than” or vice versa.) Qualitatively, the closer the plot is tothe upper left corner, the higher the overall accuracy of the test.

One convenient goal to quantify the diagnostic accuracy of a laboratorytest is to express its performance by a single number. The most commonglobal measure is the area under the ROC plot. By convention, this areais always ≧0.5 (if it is not, one can reverse the decision rule to makeit so). Values range between 1.0 (perfect separation of the test valuesof the two groups) and 0.5 (no apparent distributional differencebetween the two groups of test values). The area does not depend only ona particular portion of the plot such as the point closest to thediagonal or the sensitivity at 90% specificity, but on the entire plot.This is a quantitative, descriptive expression of how close the ROC plotis to the perfect one (area=1.0).

In a preferred embodiment the present invention relates to a method forimproving the diagnostic accuracy for rheumatoid arthritis versushealthy controls and/or patients suffering from OA by measuring in asample the concentration of at least anti-CCP and serum amyloid A andcorrelating the concentrations determined to the presence or absence ofrheumatoid arthritis, the improvement resulting in more patients beingcorrectly classified as suffering from RA versus healthy controls and/orpatients suffering from OA as compared to a classification based onanti-CCP alone. The RA marker panel comprising anti-CCP and SAA can ofcourse also be used in assessing the severity of disease for patientssuffering from RA.

As the skilled artisan will appreciate one or more additional biomarkermay be used to further improve the assessment of RA. To illustrate thisadditional potential of using anti-CCP and SAA as the key markers of apanel of markers for assessment of RA the term “at least” has been usedin the appending claims. With other words, the level measured for one ormore additional marker may be combined with the measurement of anti-CCPand SAA in the assessment of RA.

The one or more additional marker used together with anti-CCP and SAAmay be considered to be part of an RA marker panel, i.e., a series ofmarkers appropriate to further refine the assessment of RA. The totalnumber of markers in an RA marker panel is preferably less than 20markers, more preferred less than 15 markers, also preferred are lessthan 10 markers with 8 or less markers being even more preferred. Mostpreferred are RA marker panels comprising 3, 4, 5, or 6 markers intotal.

In a preferred embodiment the present invention thus relates to a methodfor assessing the absence or presence of rheumatoid arthritis in vitroby biochemical markers, comprising measuring in a sample theconcentration of anti-CCP, serum amyloid A and in addition theconcentration of one or more other marker and correlating theconcentrations of anti-CCP, SAA and of the one or more additional markerto the absence or presence of rheumatoid arthritis.

It will be appreciated that the one or more other marker may be anyknown or future marker of RA. A marker does qualify as an RA marker ifthe AUC for this marker alone, when assessing the diagnostic accuracy bycomparing patients with RA to healthy controls, is at least 0.65.

Preferably the one or more other marker is selected from the groupconsisting of C-reactive protein (=CRP), interleukin 6 (=IL-6), S100,osteopontin, RF, matrix metalloprotease 1 (=MMP-1), matrixmetalloprotease 3 (=MMP-3), hyaluronic acid, sCD14, angiogenesis markersand products of bone, cartilage or synovium metabolism.

C-reactive protein (CRP) is a homopentameric Ca²⁺-binding acute phaseprotein with 21 kDa subunits that is involved in host defense. CRPsynthesis is induced by IL-6, and indirectly by IL-1, since IL-1 cantrigger the synthesis of IL-6 by Kupffer cells in the hepatic sinusoids.The normal plasma concentration of CRP is <3 μg/ml (30 nM) in 90% of thehealthy population, and <10 μg/ml (100 nM) in 99% of healthyindividuals. Plasma CRP concentrations can, e.g. be measured byhomogeneous assay formats or ELISA. C-reactive protein is a marker forunderlying systemic inflammation.

Interleukin-6 (IL-6) is a 21 kDa secreted protein that has numerousbiological activities that can be divided into those involved inhematopoiesis and into those involved in the activation of the innateimmune response. IL-6 is an acute-phase reactant and stimulates thesynthesis of a variety of proteins, including adhesion molecules. Itsmajor function is to mediate the acute phase production of hepaticproteins, and its synthesis is induced by the cytokines IL-1 and TNF-□.IL-6 is normally produced by macrophages and T lymphocytes. The normalserum concentration of IL-6 is <5 pg/ml.

Osteopontin (=OPN) is a secreted, highly acidic, calcium-binding,phosphorylated glycoprotein. Three isoforms are known that originatefrom alternative splicing which are either free or bound to theextracellular matrix. Through a RDG-motif of the 32 kDa-peptide backboneOPN can bind to integrins such as av□3. Though it was originallypurified from bone matrix it is expressed in numerous body fluids andtissues including milk, urine, activated T-cells, macrophages,fibroblasts, smooth muscle cells, kidney tissue and some tumor cells.Its expression is stimulated in response to several cytokines, growthfactors or inflammatory mediators. Increased OPN concentrations havebeen associated with sepsis, metastatic cancer, cerebral ischemia,atherosclerotic plaques, granuloma formation in tuberculosis andautoimmune diseases such as multiple sclerosis (Chabas, D., et al.,Science 294 (2001) 1731-1735) or RA (Petrow, P. K., et al., Arthr.Rheum. 43 (2000) 1597-1605).

Rheumatoid factors (=RF) are autoantibodies directed against theconstant Fc-region of immunoglobulin G molecules (Waaler, E., ActaPathol. Microbiol. Scand. 17 (1940) 172-178; Moore, T. L., and Dorner,R. N., Clin Biochem. 26 (1993) 75-84). Though RF has some limitations itis currently the only immunologic marker of rheumatoid arthritisincluded in the ARA-criteria. Besides of RA it is also found in otherinflammatory rheumatic diseases, non-rheumatic disease and even inhealthy persons aged over 60 years (Bartfeld, H., Ann. NY Acad. Sci. 168(1969) 3040). RF autoantibodies belong to all immunoglobulin classes andmost of the assays used today do not differentiate between the isotypesIgM, IgG and IgA. These RF-assays, also termed total-RF assays,determine mostly IgM but also cover IgG or IgA to some degree dependingon the assay format and the supplier (Bas, S., et al., Ann. Rheum. Dis.61 (2002) 505-510). More recently the RF-isotypes IgG and IgA have comeinto focus for the diagnosis of RA. When all three RF-isotypes areelevated the diagnostic value of the RF-assay might be improved(Swedler, W., et al., J. Rheum. 24 (1997) 1037-1044). Additionally someprognostic value has been ascribed to certain of these RF-isotypes.Especially, a high concentration of IgA-type RF was found to be anindicator for severe disease progression (Jorgensen, C., et al., Clin.Exp. Rheum. 14 (1996) 301-304). In a marker combination according to thepresent invention the marker RF can be any form of RF-determinationincluding total RF, single specific RF-isotypes or any combination ofRF-isotypes.

The family of matrix-metalloproteinases (=MMPs) degrades almost allcomponents of the extra-cellular matrix. Hence MMPs have been related tovarious types of cancer but also to inflammatory processes in RA. MMP-1and MMP-3 are produced by fibroblasts, osteoblasts and endothelial cellsupon stimulation by pro-inflammatory cytokines like IL-1 or TNF-α.Generally MMPs are found in the circulation as inactive pro-form and themarker MMP-1 and MMP-3, respectively, as used herein also relates tosuch inactive pro-form. MMP-1 and MMP-3 have been detected in synovialfluid of RA-patients and the levels are responsive to anti-TNF-□therapy. The most preferred metalloprotease to be used in an RA markerpanel according to the present invention is MMP-1.

Instead of the metalloproteinases mentioned above it is also possible toused their corresponding inhibitors collectively referred to as tissueinhibitors of matrix metalloproteinases (=TIMPs), eg. MMP-1 and MMP-3are in vivo inactivated by TIMP-1 a sialoglycoprotein of 29.5 kD thatforms a 1:1 stoichiometric complex with the MMPs. The relation of TIMP-1and TIMP-2 to the destruction of cartilage has been investigated in RA(Ishiguro N. et al., Arthritis & Rheumatism 44 (2001) 2503-2511:“Relationship of matrix metalloproteinases and their inhibitors tocartilage proteoglycan and collagen turnover and inflammation asrevealed by analyses of synovial fluids from patients with rheumatoidarthritis”).

S100-proteins form a constantly increasing family of Ca²⁺-bindingproteins that today includes more than 20 members. The physiologicallyrelevant structure of S100-proteins is a homodimer but some can alsoform heterodimers with each other, e.g. S100A8 and S100A9. Theintracellular functions range from regulation of proteinphosphorylation, of enzyme activities, or of the dynamics of thecytoskeleton to involvement in cell proliferation and differentiation.As some S100-proteins are also released from cells, extracellularfunctions have been described as well, e.g., neuronal survival,astrocyte proliferation, induction of apoptosis and regulation ofinflammatory processes. S100A8, S100A9, the heterodimer S100A8/A9 andS100A12 have been found in inflammation with S100A8 responding tochronic inflammation, while S100A9, S100A8/A9 and S100A12 are increasedin acute inflammation. S100A8, S100A9, S100A8/A9 and S100A12 have beenlinked to different diseases with inflammatory components including somecancers, renal allocraft rejection, colitis and most importantly to RA.(Burmeister, G., and Gallacchi, G., Inflammopharmacology 3 (1995)221-230; Foell, D., et al., Rheumathology 42 (2003) 1383-1389). The mostpreferred S100 markers for use in an RA marker panel according to thepresent invention are S100A8, S100A9, S100A8/A9 heterodimer and S100A12.

CD14 is a membrane protein of pro-monocytes, monocytes, macrophages, andactivated granulocytes where it serves as a receptor forlipopolysaccharide. It induces the secretion of cytotoxic andimmunomodulating factors like reactive oxygen (0₂), tumor necrosisfactor (TNF-α), interleukins (IL-1, IL-6 and IL-8) andplatelet-activating factor (PAF). Membrane bound CD14 is shed to givesoluble CD14 (=sCD14) in response to activating or differentiatingfactors such as IFNγ or TNF-α. The physiological function of sCD14 isnot yet entirely clear. Since inflammatory and immune processes areinvolved in RA and other autoimmune diseases, sCD14 was alsoinvestigated in such diseases. When anti-CD14 therapy was evaluated as anew therapeutic option in RA previously elevated concentrations of sCD14rapidly decreased and synovitis was reduced (Horneff, G., et al., Clin.Exp. Immunol 91 (1993) 207-213).

The glycosaminoglycan hyaluronic acid is one of the macromoleculesessential for the function of a joint. It is synthesized by fibroblastsand other specialized connective tissue cells. Hyaluronic acid isinvolved in formation of the extracellular matrix and in cell to cellcontacts. High concentrations are found in synovial fluid where it isresponsible for the retention of water thereby contributing to thelubrication of joints. In rheumatoid arthritis the synthesis ofhyaluronic acid is stimulated by the proinflammatory mediators IL-1 andTNF-α leading to increased serum/plasma levels (Sawai, T., and Uzuki,M., Connective Tissue 33 (2001) 253-259).

A feature of rheumatoid arthritis is the invasion of joints withproliferating synovial tissue also known as pannus. A significant partof the pannus consists of blood vessels that supplies nutrients to thegrowing tissue. Therefore, molecules relevant in angiogenesis have beeninvestigated in RA also, both as RA markers but also as therapeutictargets (Brenchley, P. E. C., Clin. Exp. Immunol 121 (2000) 426-429).Amongst these the vascular endothelial growth factor (=VEGF) has beenevaluated in more detail. VEGF is a secreted glycoprotein that isspliced to four different isoforms. Two of these isoforms are readilydiffusible while the remaining isoforms bind tightly to heparin and aremostly found in association with heparin containing proteoglycans. VEGFacts as a chemokine on endothelial cells, monocytes and osteoblastsultimately leading to neovascularization and increased microvascularpermeability. VEGF has been detected in synovial fluid and serum of RApatients (Lee, S. S., et al., Clin. Exp. Rheumathology 19 (2001)321-324; Ballara, S., Arthritis Rheum. 44 (2001) 2055-2064). Preferably,the marker of angiogenesis is VEGF.

The most prominent joint tissues are bone, cartilage and the synovium.Since rheumatoid arthritis is a destructive disease these tissues willbe most affected. They are a likely source of potential biologicalmarkers in the field of RA. In principle these markers may come not onlyfrom the destruction of the respective tissue but also from aderegulated and/or ineffective repair process. The experienced artisanwill understand that markers of bone, cartilage or synovium metabolismcan originate either from synthesis or from destruction of thesetissues. The various markers of bone, cartilage and/or synoviummetabolism can be delineated from two different groups of proteins. Theycome either from the numerous types of collagen or from non-collagenousproteins. Non-collagenous proteins are often involved in the formationof the extracellular matrix. Some of these markers can be found in allthree tissues in varying amounts.

Markers and products of bone and/or cartilage metabolism include bothmarkers of bone and/or cartilage degradation as well as markers of boneand/or cartilage formation. Preferred markers derived from collagenmetabolism are markers such as:

-   1. Pyridinoline (=PYD), deoxy-pyridinoline (=DPD) and Glc-Gal-PYD:    Pyridinoline (=PYD) stabilizes collagen by cross-linking the strands    of the collagen triple helix. The chemical structure of PYD is very    stable and can be found in serum and urine as an end product of    collagen degradation (Knott, L., and Bailey, A. J., Bone 22 (1998)    181-187). It has been linked to arthritis (Kaufmann, J., et al.,    Rheumatology 42 (2003) 314-320). PYD monitors cartilage involvement    of joint destruction since it is released from cartilage and only to    some degree from bone while its close cousin deoxy-pyridinoline    (=DPD) originates mostly from bone. All three markers have been    linked to arthritis (Kaufmann, supra). The glycosylated form    Glc-Gal-PYD has mostly been found in synovial tissue (Gineyts, E.,    et al., Rheumatology 40 (2001) 315-323).-   2. Cross-linked telopeptides: CTX-I, CTX-II, NTX-I and the    LQ-epitope which are cross-linked telopeptides either from the C- or    N-terminus of collagens type I or type II, respectively, and of    which β-CTX-I is also known as β-CrossLaps® (Bonde, M., et al.,    Clin. Chem. 40 (1994) 2022-2025). Type I collagen carboxyterminal    telopeptide (=ICTP) refers to a fragment and marker of type I    collagen which originally has been derived from type I collagen by    cyanobromide cleavage.-   3. Linear peptides derived from collagen: The assay termed    Cartilaps® measures a linear peptide that is derived from the    C-terminal region of collagen type II.-   4. Modified amino acids: Collagen comprises modified amino acids    like hydroxyproline and galactosyl hydroxylysine which may be used    as a marker of collagen break-down (Al-Dehaimi, A. W., et al., Clin.    Chem. 45 (1999) 676-681).-   5. Collagen neoepitopes: Col2-3/4 and CIIN are neoepitopes generated    by the initial cleavage of collagen II by collagenases    (Billinghurst R. C. et al., J. Clin. Invest. 99 (1997) 1534-45).-   6. Collagen markers considered reflecting bone formation: The    N-terminal as well as the C-terminal pro-peptide of type I collagen    (=PINP and PICP), respectively, are clipped from the precursor    polypeptide (procollagen) during/after synthesis and considered    markers of bone formation. PIICP is the corresponding pro-peptide    from collagen type II, whereas PIIINP is derived from collagen III.

Preferably the marker of bone and/or cartilage metabolism also my be anon-collagenous marker, like: CS846, which is a chondriotin sulfateepitope created during aggrecan synthesis; cartilage oligomeric matrixprotein (=COMP) that has bridging functions in cartilage (Saxne, T., andHeinegard, D., Br. J. Rheumatol. 31 (1992) 583-591); cartilageintermediate layer protein (=CILP), which is a matrix protein ofcartilage (Lorenzo, P., et al., J. Biol. Chem. 273 (1998) 23463-23468);cartilage matrix proteins 1-3 also known as matrilins; chondromodulinsthat act as signaling molecules in cartilage (Suzuki, F., Connect.Tissue Res. 35 (1996) 303-307); cartilage derived retinoicacid-sensitive protein (=CD-RAP) or MIA, which has a yet to be definedfunction in chondrocyte modulation (Müller-Ladner, U., et al.,Rheumatology 38 (1999) 148-154); osteocalcin, which is synthesized byosteoblasts, belongs to the major non-collagen matrix protein of boneand is used to monitor bone turnover (Gundberg, C. M., et al., J. Clin.Ligand Assay 21 (1998) 128-138); and the bone sialoproteins, which aremajor non-collagen matrix proteins of bone, such as bone sialoproteinII, now known as bone sialoprotein, which e.g., has been evaluated asmarker for bone turn-over (Saxne, T., et al., Arthr. Rheum. 38 (1995)82-90).

Products of metabolism within the synovium which may be used as a markerin assessing RA include: CTX-III, which is a telopeptide derived fromcollagen type III, YKL40 the later being a chitinase 3 like protein ofthe extracellular matrix (Johansen, J. S., et al., Scand. J. Rheumatol.30 (2001) 297-304), and aggrecan, which is a building block ofproteoglycans as well as its degradation product keratan sulfate.

Preferably the RA marker panel comprises at least three markers, whereinanti-CCP, SAA and a third marker selected from the group consisting ofCRP, IL-6, S100, osteopontin, RF, MMP-1, MMP-3, hyaluronic acid, and aproduct of collagen metabolism are contained.

In the assessment of RA a marker panel comprising anti-CCP, SAA andS100, especially, S100A12 is preferred.

As mentioned further above (see ARA criteria)—despite severelimitations—the rheumatoid factor (RF) currently is the only biochemicalmarker generally accepted to aid in establishing the diagnosis of RA. Itis clearly expected that the marker combination of the present inventionwill significantly improve the diagnosis of RA and will supplement ormight be even finally replace the RF assay. The use of a marker panelcomprising at least anti-CCP and serum amyloid A in the diagnosis of RAtherefore represents a further preferred embodiment of the presentinvention.

As the skilled artisan will appreciate one or more additional marker maybe used to further improve the diagnostic accuracy, or, where requiredincrease the diagnostic sensitivity at the expense of specificity orvice versa. In some diagnostic areas, e.g., in the detection of anHIV-infection sensitivity is of utmost importance. The high sensitivityrequired may be achieved at the expense of specificity, leading to anincreased number of false positive cases. In other cases, e.g. as asimple example, when assessing blood group antigens, specificity is ofparamount importance.

A further preferred embodiment relates to the use of a marker panel inthe diagnosis of RA the panel comprising anti-CCP, serum amyloid A andat least one additional marker selected from the group consisting ofCRP, IL-6, S100, osteopontin, RF, MMP-1, MMP-3, hyaluronic acid, sCD14,angiogenesis markers and products of bone, cartilage or synoviummetabolism.

It is obvious that the method according to the present invention willalso be of great use in assessing the severity of RA. The higher thelevel of anti-CCP and/or the higher the level of SAA the more severe isthe disease. With the marker combination or marker panels now at hand itwill be no more than routine experimentation to develop e.g., diseasescores as an indicator for severity of disease. The method according tothe present invention thus is preferably also used to assess theseverity of disease.

Beyond doubt the method of the present invention will also be of greathelp in monitoring the course of disease. This is most easily achievedby measuring in a patient sample anti-CCP and SAA as well as optionallyadditional markers at various points in time and comparing the absoluteand/or the relative levels of the markers at these different timepoints. It thus is further preferred to use the method according to thepresent invention to monitor the course of disease in a patient with RA.

It is also recognized that the present invention will be of great helpin assessing the efficacy of any treatment for RA. The efficacy oftreatment will be reflected by changes in the marker level. If atreatment has the desired effect at least one of the two marker levelsof anti-CCP or SAA will decrease. The method according to the presentinvention thus preferably is also used to assess the efficacy oftreatment. The same phenomenon, i.e. a reduction in marker level of atleast one of anti-CCP or SAA can easily be applied for selection of theright drug as well as the most appropriate dosing of drugs in RA. Theuse of a method of this invention in selection of the right drug and/orthe most appropriate dosing is also preferred.

The method of the present invention will also enable the selection andidentification of new drugs in the field of RA. This applicationrepresents a further preferred embodiment.

It will also be a great advantage that sub-groups of patients can now beidentified for and in clinical studies which differ in their level ofanti-CCP and SAA and to correlate this difference in marker level to theefficacy of the drug under investigation.

The present invention also relates to a kit for performing the method ofthis invention comprising the reagents required to specifically measureanti-CCP and serum amyloid A, respectively. The kit may optionallycomprise auxiliary reagents for performing the measurement of bothanti-CCP and SAA.

The examples and figures herein are provided to aid the understanding ofthe present invention, the true scope of which is set forth in theappended claims. It is understood that modifications can be made in theprocedures set forth without departing from the spirit of the invention.

SPECIFIC EMBODIMENTS Example 1 Study Population

Samples derived from 389 highly characterized RA patients with maximumdisease duration of 15 years were collected in five European centerswith a follow-up of two years. All individuals were diagnosed asRA-patients according to the ARA-criteria and had a functional status of≦III as classified by the ARA classification criteria (Hochberg, M. C.,et al., Arthritis Rheum. 35 (1992) 498-502). All patients weredocumented with an extensive case report form (=CRF). The CRF includedthe Health Assessment Questionnaire, the SF36 Questionnaire, swollen andtender joint count, the Larsen Score, laboratory parameters, clinicalhistory of relevant surgery, medication, co-morbidities and medicationfor co-morbidities. X-rays were taken every year following astandardized procedure. Only baseline samples obtained from the subjectsincluded in this study were included in the present analysis.

Samples derived from 624 control subjects were collected as well. Fromthese controls only RA-positive subjects but not other forms ofarthritis were excluded. 200 samples were drawn from this cohort toage-match the RA-samples of the study. Since the focus of the study wasto discriminate RA not only from healthy subjects but also from otherjoint diseases, 203 patients with either tibiofemoral or patellofemoralOA of the knee were added as disease controls. For these OA patientsclinical and laboratory parameters were determined and radiographicKellgren & Lawrence Score was calculated (Kellgren, J. H., and Lawrence,J. S., Ann. Rheum. Dis. 16 (1957) 494-502).

Demographic data for the study population are given in Table 1.

TABLE 1 Patient collectives Collective N Age Gender (f/m) RA 389 59.1(16-83) 256/133 Controls incl. OA 403 60.7 (38-92) 201/203

Example 2 Markers Measured

Table 2 presents the assays used and gives the test format as well asthe suppliers of the assays. Most of the assays were manual microtiterplate format (=MTP) ELISAs. RF and CRP were determined in a homogeneoustest format on an automatic Hitachi analyzer. Marker concentrations weredetermined in serum samples with these commercially available assays forpatients as well as for controls.

TABLE 2 Assays and Suppliers Biomarker Assay type/format Source Anti-CCPSandwich ELISA, MTP Axis-Shield, Dundee (UK) CRP Homogenous assay,Hitachi Roche Diagnostics, Mannheim (FRG) Hyaluronic Sandwich ELISA, MTPChugai, Tokyo (J) acid RF Homogenous assay, Hitachi Roche Diagnostics,Mannheim (FRG) SAA SAA Sandwich ELISA, MTP Biosource, Nivelles (B)

Example 3 Statistical Evaluation

The patient cohorts were randomly split in a training set (app. 67%) andin a test set (app. 33%). On the training set a classification algorithmwas developed and on the independent test set the algorithm wasvalidated. As can be seen in Table 3 the respective sets were closelymatched in size as well as in age.

TABLE 3 Age distribution of collectives Group Study N Mean Max q3 Medianq1 Min Training RA 259 58.7 87 68 59 51 23 Training Controls 273 60.5 9270 61 51 42 incl. OA Test RA 130 59.8 83 68 61 52 16 Test Controls 13061.2 84 70 62.5 51 38 incl. OA

The classification algorithms were generated with the RegularizedDiscriminant Analysis (RDA), which is a generalization of the commonDiscriminant Analysis, i.e. Quadratic- and Linear Discriminant Analysis(McLachlan, G. J., Discriminant Analysis and Statistical PatternRecognition, Wiley Series in probability and mathematical statistics,1992). In the RDA alternatives to the usual maximum likelihood (plug-in)estimates for the covariance matrices are used. These alternatives arecharacterized by two parameters (λ, γ), the values of which arecustomized to individual situations by jointly minimizing a sample-basedestimate of future misclassification risk (Friedman, J. H., RegularizedDiscriminant Analysis, Journal of the American Statistical Association84 (1989) 165-175). As an alternative method Support Vector Machinesalgorithms (Hastie, Trevor, Tibshirani, Robert, Friedman, Jerome, TheElements of Statistical Learning, Springer Series in Statistics, 2001)can be fitted with comparable classification results.

The marker panels were stepwise constructed starting from the bestsingle marker for the classification problem and ending when the totalclassification error does not change remarkable any more. In order togain centralized distributions every single marker was transformed withthe natural logarithmic function. 10-fold cross validation was used onthe training set to get robust estimates of the total error(sensitivity, specificity). Once the marker panel was defined, it wasvalidated without any further adjustment with an independent test set.

Example 4 Identification of a Marker Panel for the Diagnosis of RA

Table 4 presents the classification results of patients diagnosed withRA versus controls incl. OA on the training set. The first markerselected was anti-CCP, the second one SAA and the third and last onehyaluronic acid when the algorithm stopped. As a reference theclassification results for total RF are presented, which as mentionedabove is currently the only biochemical marker forming part of theARA-criteria.

The aim of the current invention was to improve the correct diagnosis ofRA versus controls including OA. The diagnostic value of the identifiedmarker panel is best reflected in Table 4 by the total error of theclassification. RF, currently the single biological marker included inthe ARA-criteria, gives a total error of 0.18. The preferred combinationof anti-CCP and SAA improves the classification with a total error of0.14. Adding a third marker finally helps to further minimize themisclassification. The marker panel anti-CCP, SAA plus hyaluronic acidhas a total error of 0.13.

The ROC-curves for the markers and marker combinations, respectively, ofTable 4 are shown in FIGS. 1 to 4.

TABLE 4 Classification results on the training set of patients diagnosedwith RA versus controls incl. OA Cross validation (10 fold) No of Markeror Method TOTAL Markers marker panel (RDA) ERROR Sensitivity Specificity1 log total RF λ = 0, γ = 0 0.18232 68.2% 95.4% 1 log anti-CCP λ = 0, γ= 0 0.14106 74.7% 96.7% 2 log anti-CCP, λ = 0, γ = 0 0.14095 75.4% 96.4%log SAA 3 log anti-CCP, λ = 0, 0.13166 76.7% 96.8% log SAA, log γ = 0.25hyaluronic acid

Most critical for the approach chosen in this study is the question ifit has a general applicability. To test this, the marker panelidentified in the training set was validated with an independent testset. As the skilled artisan will understand, the results of the trainingand the test set may differ slightly because both sets were trulyindependent. Table 5 gives the classification results using the samesingle markers or marker panels as in Table 4. As in the training setthe combination of anti-CCP and SAA, and optionally hyaluronic acidreduces the total error of the classification. The results presented inTable 4 and 5 clearly show that the combination of anti-CCP and SAA, andoptionally at least one additional marker significantly improves thediagnosis of RA especially as compared to total RF.

TABLE 5 Classification results on the test set of patients diagnosedwith RA versus controls including patients with OA Classification ofTest Set No of Marker or Method TOTAL Markers marker panel (RDA) ERRORSensitivity Specificity 1 log total RF λ = 0, γ = 0 0.19615 66.2% 94.6% 1 log anti-CCP λ = 0, γ = 0 0.16538 66.9% 100% 2 log anti-CCP, λ = 0, γ= 0 0.12692 74.6% 100% log SAA 3 log anti-CCP, λ = 0, 0.13077 73.9% 100%log SAA, log γ = 0.25 hyaluronic acid

1. A method for assessing an absence or presence of rheumatoid arthritisin a patient comprising measuring in a sample from the patient aconcentration of anti-CCP and a concentration of serum amyloid A,wherein the sample is blood, plasma, or serum, and combining theconcentrations determined in the measuring step to obtain a combinedvalue and comparing the combined value to a cut-off value establishedfrom a reference population wherein a value below the cut-off value isindicative of the absence of rheumatoid arthritis in the patient and avalue above the cut-off is indicative of the presence of rheumatoidarthritis in the patient.
 2. A method for assessing an absence orpresence of rheumatoid arthritis in a patient comprising measuring in asample from the patient, wherein the sample is blood, plasma, or serum,a concentration of anti-CCP, a concentration of serum amyloid A, and aconcentration of a marker selected from the group consisting ofC-reactive protein (CRP), interleukin 6 (IL-6), S100, osteopontin,rheumatoid factor (RF), matrix metalloprotease 1 (MMP-1), matrixmetalloprotease 3 (MMP-3), hyaluronic acid, and sCD14, combining theconcentrations determined in the measuring step to obtain a combinedvalue, and comparing the combined value to a cut-off value establishedfrom a reference population to assess the absence or presence ofrheumatoid arthritis in the patient, wherein a combined value below thecut-off value is indicative of absence of rheumatoid arthritis and acombined value above the cut-off value is indicative of presence ofrheumatoid arthritis.
 3. The method of claim 2 wherein the selectedmarker is hyaluronic acid.
 4. The method according to claim 1 whereinthe cut-off value is established by analyzing a receiver operatingcharacteristic curve derived from the reference population.
 5. Themethod according to claim 2 wherein the cut-off value is established byanalyzing a receiver operating characteristic curve derived from thereference population.